Method for determining b vitamins and their vitamers

ABSTRACT

The present disclosure discusses a method of separating a sample (e.g., B vitamins and their vitamers) including coating a flow path of a chromatographic system; injecting the sample into the chromatographic system; flowing the sample through the chromatographic system; separating the sample; and analyzing the separated sample. In some examples, the coating applied to the surfaces defining the flow path is non-binding with respect to the sample—and the separated sample. Consequently, the sample does not bind to the low-binding surface of the coating (e.g., organosilica coating) of the flow path. The applied coating provides an inert barrier that is beneficial for B vitamin analysis, including improved peak shape (less tailing and narrower peak), high sensitivity, and no carry-over.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalPatent Application No. 63/132,558, filed Dec. 31, 2020, whichapplication is hereby incorporated by reference in its entirety, withany definitions of terms in the present application controlling.

FIELD OF THE TECHNOLOGY

The present disclosure relates to methods for determining B vitamins andtheir vitamers. More specifically, this technology relates to analyzingB vitamins and their vitamers via a liquid chromatography (LC) systemand column that have been modified with a layer of inert material, forexample the LC system and column's internal metal surface has beenmodified with a deposited inert material.

BACKGROUND

Liquid chromatography (LC) is an analytical separation technique, whichenables the separation of a mixture of chemical species on the basis ofdifferential interactions between the compounds of the mixture and astationary phase—defined as primary interactions, which are theanticipated interactions between the mixture, the designed stationaryphase and the modulations from specifically chosen mobilephase/environmental conditions. These interactions are dependent on anumber of controlled variables, such as mobile phase composition,temperature and flow rate.

SUMMARY

Strong interactions between certain analytes and metal surfaces inliquid chromatography flow path may result in poor chromatographic peakshapes, severe analyte losses, inconsistent peak responses, and otherissues in LC, which lead to inaccurate results. To reduce the impact ofthese interactions, workarounds or additional steps in the methods areoften required, which adds complexity to the methods and reduces thelaboratory productivity.

Ongoing efforts to reduce chelation and secondary chromatographicinteractions of analytes with metal chromatographic surfaces in aneffort to facilitate chromatographic separation having higherresolutions are therefore needed. In addition, variability in theseparation and detection of compounds can be caused by many factors. Onesuch factor is analyte/surface interactions of compounds with theanalytical column. Such interactions can be problematic, especially atvery low concentrations of analytes. This is true for B vitamins andtheir vitamers.

Secondary interaction or adsorption of metal sensitive analytes toactive sites dispersed throughout the metallic surface in liquidchromatography based separations have often been challenging toseparate. Metal-ion mediated adsorption in liquid chromatography (LC)has been observed as a contributing factor to poor peak shape, tailing,and diminished recovery of sensitive analytes. To address problemsexperienced in separations in metallic fluidic systems, column hardwareusing a coating has been developed to define a low-binding surface(s)(LBS).

Using LBS in LC can provide an effective solution to mitigate analyteinteractions with metal surfaces. One example is using LBS to analyzeB-group vitamins by liquid chromatography, coupled with tandemquadrupole mass spectrometry (LC-MS/MS). B vitamins are essentialmicronutrients for normal human metabolic and physiological functions.They cannot be synthesized in vivo in sufficient amounts to meetphysiological requirements. Abundant supply of vitamins in diet isimportant for the growth and health of human body. B vitamins are oftenenriched or fortified in foods. Determination of B vitamins in foods iscommonly required in the food manufacturing process. Systems with LBScan enhance LC performance when compared to a conventional system andcolumn. Better peak shapes, increased response, higher sensitivity andno analyte carry-over are some of the potential benefits when using LBS.

For example, using LBS in LC system and columns with B vitamins andtheir vitamers can help increase the peak area (intensity), reduce peaktailing and peak width for B vitamins (peak shape), which can result ina better response factors and detection sensitivity. The use of LBS inLC systems and columns can also help reduce the potential carry-overissue for B vitamins. And, after extensive use (such as 170 injections),a LBS configuration compared to configuration with no LBS can stillexhibit higher response factors (>150%) for some B vitamins, such asthiamine, biotin, nicotinic acid, and 5MTHF.

The benefits of using LBS with B vitamins varies from compound tocompound. Some of the B vitamin compounds that experience a benefitassociated with LBS systems include flavin mononucleotide, thiamine,thiamine pyrophosphate, pyridoxal 5′-phosphate and pantothenic acid.

No negative impact associated with using LBS has been observed for any Bvitamin. Specifically, LBS coated hardware does not appear to adverselyaffect chromatographic performance or recovery of B vitamins. Forexample, as discussed herein, after extensive use (such as 170injections), a LBS configuration compared to configuration with no LBScan still exhibit higher response factors (>150%) for some analytes suchas B vitamins and their vitamers.

In addition, sample throughput can be increased by using the technologyof the present disclosure. Sample throughput can be increased by reducedpeak tailing and increased resolution. For example, if impurities areclosely eluting with the native peak and the native peak was exhibitinga degree of tailing, a user (e.g., an analyst) may try to extend thegradient or run-time to resolve impurities to an acceptable resolutionbetween peaks that facilitated accurate quantitation. In the absence oftailing, a user could shorten the run time by using a steeper slope inthe gradient. This could effectively elute everything faster and closertogether. But the resolution between peaks, while decreasing, may stillbe sufficient for the analytes of interest since tailing is not presentto interfere with integration or cause a co-elution. With reduced peaktailing, new trace species can be detected by being able to see peaksthat were formerly covered by peak tailing.

Increased resolution or more time between peaks can allow a user to runfaster methods with increased throughput. If resolution has increased,then peak capacity increases meaning more peaks can fit in the samechromatogram or a faster separation could be run at the cost ofresolution and peak capacity if the critical pair of interest wereresolved sufficiently to start with.

A chromatographic column incorporating the coating of the presentdisclosure has been designed to minimize negative analyte/surfaceinteractions for compounds, such as compounds with analytes including Bvitamins and their vitamers. Existing techniques to mitigate theseinteractions, such as system passivation with nitric acid, are timeconsuming and only produce temporary performance gains. It is difficultto determine when the system is fully passivated and ready to operate.If attempts are made to obtain data for quantitative studies before fullpassivation is reached, the lower end of the calibration curve would notbe detected because the analyte still has metallic surfaces it can bindto.

A LBS, such as a surface with an alkylsilyl coating, on the surface areaof the flow path of a chromatographic system can minimize theinteractions between B vitamins (including their vitamers) and themetallic surfaces of chromatographic flow paths (e.g., column interiorwalls, interior of tubing, surfaces of frits, sample injectors, etc.).Consequently, the coated metallic surfaces improve liquid chromatographyseparations for B vitamins and their vitamers. The use of alkylsilylcoatings on metal flow paths allows the use of metal chromatographicflow paths, which are able to withstand high pressures at fast flowrates, high pressure generated using stationary phases with smallparticles (which can be slow flow as well), and high pressure generatedfrom longer column beds, while minimizing the secondary chromatographicinteractions between B vitamin compounds and the metal. These componentsmade of high-pressure material and modified with a coating can betailored so that the internal flow paths reduce secondarychromatographic interactions. The coating covers the metallic surfacesthat are exposed to the fluidic path.

In one aspect, the present disclosure is directed to a method ofseparating and analyzing a metal-sensitive sample. The method includesinjecting the metal-sensitive sample into a chromatographic system,wherein the chromatographic system includes a metallic flow path with alow-bind surface coating; flowing the metal-sensitive sample through thechromatographic system; separating the metal-sensitive sample, whereinthe metal-sensitive sample includes one or more B vitamin and/or Bvitamin vitamer; and analyzing the separated sample by passing theseparated metal-sensitive sample through a mass spectrometer. In someembodiments, the sample includes two or more of B vitamins and/or Bvitamin vitamers, and analyzing the separated sample includessimultaneously analyzing the two or more of B vitamins and/or B vitaminvitamers to determine the B vitamins and/or B vitamin vitamers presentin the sample. In some embodiments, the method includes, prior toinjecting the metal-sensitive sample into the chromatographic system,extracting the metal-sensitive sample from a sample matrix or samplematrices. In some embodiments extracting the metal-sensitive sample froma sample matrix or sample matrices includes separate sample preparationsfor different B vitamins. In some embodiments, flowing themetal-sensitive sample through the chromatographic system includes usinga single liquid chromatography method to determine the B vitamins and/orB vitamin vitamers. In some embodiments, using the single liquidchromatography method includes using liquid chromatography-massspectrometry (LC/MS). In some embodiments, the liquidchromatography-mass spectrometry (LC/MS) is a tandem quadrupole massspectrometer (LC-MS/MS). In some embodiments, the B vitamins or Bvitamin vitamers include at least one of riboflavin (B₂), flavinmononucleotide (FMN), biotin, nicotinic acid (B₃), nicotinamide (B₃),nicotinamide adenine dinucleotide (NAD), pyridoxine (B₆), pyridoxal(B₆), pyridoxal 5′-phosphate (PLP), thiamine (B₁), thiaminepyrophosphate (TPP), folic acid (B₉), pantothenic acid (B₅), or5-methyl-tetrahydrofolate (5MTHF). In some embodiments, the sample is adietary supplement or an energy drink.

In one aspect, the present disclosure is directed a method of separatingand analyzing a metal-sensitive sample. The method includes injectingthe metal-sensitive sample into a chromatographic system, wherein thechromatographic system includes a metallic flow path with a coating;flowing the metal-sensitive sample through the chromatographic system;separating the metal-sensitive sample, wherein the metal-sensitivesample includes two or more B vitamins and/or B vitamin vitamers; andsimultaneously analyzing the two or more B vitamins and/or B vitaminvitamers of the separated sample. In some embodiments, the coating is alow-bind surface coating. In some embodiments, analyzing the separatedsample includes passing the separated metal-sensitive sample through amass spectrometer. In some embodiments, the method includes, prior toinjecting the metal-sensitive sample into the chromatographic system,extracting the metal-sensitive sample from a sample matrix or samplematrices. In some embodiments, the method of extracting themetal-sensitive sample from a sample matrix or sample matrices includesseparate sample preparations for different B vitamins and/or B vitaminvitamers. In some embodiments, flowing the metal-sensitive samplethrough the chromatographic system includes using a single liquidchromatography method to determine the B vitamins and/or B vitaminvitamers. In some embodiments, the single liquid chromatography methodincludes using liquid chromatography-mass spectrometry (LC/MS). In someembodiments, the liquid chromatography-mass spectrometry (LC/MS) is atandem quadrupole mass spectrometer (LC-MS/MS). In some embodiments, theB vitamins or B vitamin vitamers include riboflavin (B₂), flavinmononucleotide (FMN), biotin, nicotinic acid (B₃), nicotinamide (B₃),nicotinamide adenine dinucleotide (NAD), pyridoxine (B₆), pyridoxal(B₆), pyridoxal 5′-phosphate (PLP), thiamine (B₁), thiaminepyrophosphate (TPP), folic acid (B₉), pantothenic acid (B₅), or5-methyl-tetrahydrofolate (5MTHF). In some embodiments, the sample is adietary supplement or an energy drink.

The above aspects and features of the present technology providenumerous advantages over the prior art. In some embodiments, there arenumerous benefits incorporating the coating on the column. For example,the present disclosure shows improvement in the LC column and systemperformance of peak intensity, peak shape, carry-over, and responsefactor. The present disclosure includes methods for determining Bvitamins in energy drinks and dietary supplements.

BRIEF DESCRIPTION OF THE DRAWINGS

The technology will be more fully understood from the following detaileddescription taken in conjunction with the accompanying drawings, inwhich:

FIG. 1 is a schematic of a chromatographic flow system including achromatography column and various other components, in accordance withan illustrative embodiment of the technology. A fluid is carried throughthe chromatographic flow system with a fluidic flow path extending froma fluid manager to a detector, such as a MS detector.

FIG. 2 is a flow chart of a method of coating a fluidic path (such as afluidic path in a chromatography system) according to an illustrativeembodiment of the technology.

FIG. 3 is a flow chart showing a method of tailoring a fluidic flow pathfor separation of a sample including a B vitamin and/or B vitaminvitamer, in accordance with an illustrative embodiment of thetechnology.

FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, FIG. 4F, FIF. 4G, FIG. 4H,FIG. 4I, FIG. 4J, FIG. 4K, FIG. 4L, FIG. 4M, FIG. 4N, and FIG. 4Odisplay structures of selected B vitamins. FIG. 4A, FIG. 4B, FIG. 4C,FIG. 4D, FIG. 4E, FIG. 4F, FIG. 4G, FIG. 4H, FIG. 4I, FIG. 4J, FIG. 4K,FIG. 4L, FIG. 4M, FIG. 4N, and FIG. 4O display structures of vitaminB₁₂, riboflavin (B₂), biotin, FMN (flavin mononucleotide), nicotinicacid (B₃), nicotinamide (B₃), nicotinamide adenine dinucleotide,pyridoxine (B₆), pyridoxamine (B₆), pyridoxal (B₆), PLP (Pyridoxal5′-phosphate), thiamine (B₁), folic acid (B₉), 5MTHF(5-methyl-tetrahydrofolate), TPP (Thiamine pyrophosphate), andpantothenic acid (B₅), respectively.

FIG. 5 displays the liquid chromatography conditions in the Associationof Official Analytical Chemists (AOAC) Official Method for simultaneousdetermination of total vitamins B₁, B₂, B₃, and B₆ and other nutrientsin matrices, such as infant formula and related nutritionals.

FIG. 6A and FIG. 6B display the multiple reaction monitoring (MRM)transitions and MS detection parameters for 18 vitamins, including theadditional vitamins B₅, B₉, B₁₂ and biotin, that were used for the Xevo™TQ-S Micro MS System.

FIG. 7 displays a table of calibration data, in accordance with thepresent disclosure.

FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D, FIG. 8E, FIG. 8F, FIG. 8G, FIG. 8H,FIG. 8I, FIG. 8J, FIG. 8K, FIG. 8L, FIG. 8M, FIG. 8N, FIG. 8O, FIG. 8P,FIG. 8Q and FIG. 8R show comparison plots of the vitamin peak areas from7 replicate injections of a standard mix solution (at a concentration of1 μg/mL) in the SOP configuration and the LBS configuration. FIG. 8A,FIG. 8B, FIG. 8C, FIG. 8D, FIG. 8E, FIG. 8F, FIG. 8G, FIG. 8H, FIG. 8I,FIG. 8J, FIG. 8K, FIG. 8L, FIG. 8M, FIG. 8N, FIG. 8O, FIG. 8P, FIG. 8Qand FIG. 8R display peak areas for pyridoxine, thiamine, MeB12(methyl-cobalamine), TPP (Thiamine pyrophosphate), Aden B12(adenosyl-cobalamin), nicotinic acid, riboflavin, nicotinamide, FMN(flavin mononucleotide), nicotinamide adenine dinucleotide (NAD), B₁₂,folic acid, pyridoxamine, 5MTHF (5-methyl-tetrahydrofolate), pyridoxal,pantothenic acid, PLP (Pyridoxal 5′-phosphate), and biotin,respectively, in accordance with the present disclosure.

FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E, FIG. 9F, FIG. 9G, and FIG.9H compare the chromatograms for the LBS configuration and the SOPconfiguration for flavin mononucleotide (FMN), thiamine (B₁), pyridoxal5′-phosphate (PLP), and pantothenic acid (B₅), respectively, inaccordance with the present disclosure. FIG. 9A, FIG. 9C, FIG. 9E, andFIG. 9G display the chromatograms for the LBS configuration for flavinmononucleotide (FMN), thiamine (B₁), pyridoxal 5′-phosphate (PLP), andpantothenic acid (B₅), respectively, in accordance with the presentdisclosure. FIG. 9B, FIG. 9D, FIG. 9F, and FIG. 9H display thechromatograms for the SOP configuration for flavin mononucleotide (FMN),thiamine (B₁), pyridoxal 5′-phosphate (PLP), and pantothenic acid (B₅),respectively, in accordance with the present disclosure.

FIG. 10A, FIG. 10B, FIG. 10C, and FIG. 10D are comparisons of LC-MSchromatograms of FMN (FIG. 10A), Thiamine (FIG. 10B), PLP (FIG. 10C),and Pantothenic acid (FIG. 10D) from the initial injection of in thesame standard solution using LBS surfaces (LBS configuration) vsstandard surfaces (SOP configuration), in accordance with the presentdisclosure.

FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, FIG. 11E, FIG. 11F, FIG. 11G,FIG. 11H, FIG. 11I, FIG. 11J, FIG. 11K, FIG. 11L, FIG. 11M, FIG. 11Ncompare thiamine pyrophosphate (TPP) chromatograms from initial seveninjections for the SOP configuration and the LBS configuration, inaccordance with the present disclosure. FIG. 11A, FIG. 11B, FIG. 11C,FIG. 11D, FIG. 11E, FIG. 11F, FIG. 11G display chromatograms from7^(th), 6^(th), 5^(th), 4^(th), 3^(rd), 2^(nd) and 1^(st) injections forthe SOP configuration, respectively. FIG. 11H, FIG. 11I, FIG. 11J, FIG.11K, FIG. 11L, FIG. 11M, FIG. 11N display chromatograms from 7^(th),6^(th), 5^(th), 4^(th), 3^(rd), 2^(nd) and 1^(st) injections for the LBSconfiguration.

FIG. 12A, FIG. 12B, FIG. 12C, FIG. 12D, FIG. 12E, FIG. 12F, FIG. 12G,FIG. 12H, FIG. 12I, FIG. 12J, FIG. 12K, FIG. 12L, FIG. 12M, FIG. 12N,FIG. 12O and FIG. 12P compare the chromatograms of flavin mononucleotide(FMN) in standard solutions for the SOP configuration and the LBSconfiguration, in accordance with the present disclosure. FIG. 12A, FIG.12B, FIG. 12C, FIG. 12D, FIG. 12E, FIG. 12F, FIG. 12G, FIG. 12H displaychromatograms for 10 ppm, 3 ppm, 1000 ppb, 300 ppb, 100 ppb, 30 ppb, 10ppb, and 3 ppb of flavin mononucleotide (FMN) concentrations,respectively, in standard solutions for the SOP configuration. FIG. 12I,FIG. 12J, FIG. 12K, FIG. 12L, FIG. 12M, FIG. 12N, FIG. 12O and FIG. 12Pdisplay chromatograms for 10 ppm, 3 ppm, 1000 ppb, 300 ppb, 100 ppb, 30ppb, 10 ppb, and 3 ppb of flavin mononucleotide (FMN) concentrations,respectively, in standard solutions for the LBS configuration.

FIG. 13A, FIG. 13B, FIG. 13C, FIG. 13D, FIG. 13E, FIG. 13F, FIG. 13G,FIG. 13H, FIG. 13I, FIG. 13J, FIG. 13K, FIG. 13L, FIG. 13M, FIG. 13N,FIG. 13O and FIG. 13P compare the chromatograms of pyridoxal5′-phosphate (PLP) in standard solutions for the SOP configuration andthe LBS configuration, in accordance with the present disclosure. FIG.13A, FIG. 13B, FIG. 13C, FIG. 13D, FIG. 13E, FIG. 13F, FIG. 13G and FIG.13H display chromatograms for 10 ppm, 3 ppm, 1000 ppb, 300 ppb, 100 ppb,30 ppb, 10 ppb, and 3 ppb of pyridoxal 5′-phosphate (PLP)concentrations, respectively, in standard solutions for the SOPconfiguration. FIG. 13I, FIG. 13J, FIG. 13K, FIG. 13L, FIG. 13M, FIG.13N, FIG. 13O and FIG. 13P display chromatograms for 10 ppm, 3 ppm, 1000ppb, 300 ppb, 100 ppb, 30 ppb, 10 ppb, and 3 ppb of pyridoxal5′-phosphate (PLP) concentrations, respectively, in standard solutionsfor the LBS configuration.

FIG. 14A, FIG. 14B, FIG. 14C, FIG. 14D, FIG. 14E, FIG. 14F, FIG. 14G,FIG. 14H, FIG. 14I, FIG. 14J, FIG. 14K, FIG. 14L, FIG. 14M, FIG. 14N,FIG. 14O and FIG. 14P compare the chromatograms of thiamine in standardsolutions for the SOP configuration and the LBS configuration, inaccordance with the present disclosure. FIG. 14A, FIG. 14B, FIG. 14C,FIG. 14D, FIG. 14E, FIG. 14F, FIG. 14G and FIG. 14H displaychromatograms for 10 ppm, 3 ppm, 1000 ppb, 300 ppb, 100 ppb, 30 ppb, 10ppb, and 3 ppb of thiamine concentrations, respectively, in standardsolutions for the SOP configuration. FIG. 14I, FIG. 14J, FIG. 14K, FIG.14L, FIG. 14M, FIG. 14N, FIG. 14O and FIG. 14P display chromatograms for10 ppm, 3 ppm, 1000 ppb, 300 ppb, 100 ppb, 30 ppb, 10 ppb, and 3 ppb ofthiamine concentrations, respectively, in standard solutions for the LBSconfiguration.

FIG. 15A, FIG. 15B, FIG. 15C, FIG. 15D, FIG. 15E, FIG. 15F, FIG. 15G,FIG. 15H, FIG. 15I, FIG. 15J, FIG. 15K, FIG. 15L, FIG. 15M, FIG. 15N,FIG. 15O and FIG. 15P compare the chromatograms of pantothenic acid instandard solutions for the SOP configuration and the LBS configuration,in accordance with the present disclosure. FIG. 15A, FIG. 15B, FIG. 15C,FIG. 15D, FIG. 15E, FIG. 15F, FIG. 15G and FIG. 15H displaychromatograms for 10 ppm, 3 ppm, 1000 ppb, 300 ppb, 100 ppb, 30 ppb, 10ppb, and 3 ppb of pantothenic acid concentrations, respectively, instandard solutions for the SOP configuration. FIG. 15I, FIG. 15J, FIG.15K, FIG. 15L, FIG. 15M, FIG. 15N, FIG. 15O and FIG. 15P displaychromatograms for 10 ppm, 3 ppm, 1000 ppb, 300 ppb, 100 ppb, 30 ppb, 10ppb, and 3 ppb of pantothenic acid concentrations, respectively, instandard solutions for the LBS configuration.

FIG. 16A, FIG. 16B, FIG. 16C, FIG. 16D, FIG. 16E, FIG. 16F, FIG. 16G,FIG. 16H, FIG. 16I, FIG. 16J, FIG. 16K, FIG. 16L, FIG. 16M, FIG. 16N,FIG. 16O and FIG. 16P compare the chromatograms of nicotinic acid instandard solutions for the SOP configuration and the LBS configuration,in accordance with the present disclosure. FIG. 16A, FIG. 16B, FIG. 16C,FIG. 16D, FIG. 16E, FIG. 16F, FIG. 16G and FIG. 16H displaychromatograms for 10 ppm, 3 ppm, 1000 ppb, 300 ppb, 100 ppb, 30 ppb, 10ppb, and 3 ppb of nicotinic acid concentrations, respectively, instandard solutions for the SOP configuration. FIG. 16I, FIG. 16J, FIG.16K, FIG. 16L, FIG. 16M, FIG. 16N, FIG. 16O and FIG. 16P displaychromatograms for 10 ppm, 3 ppm, 1000 ppb, 300 ppb, 100 ppb, 30 ppb, 10ppb, and 3 ppb of nicotinic acid concentrations, respectively, instandard solutions for the LBS configuration.

FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D, FIG. 17E, FIG. 17F, FIG. 17G,FIG. 17H, FIG. 17I, FIG. 17J, FIG. 17K, FIG. 17L, FIG. 17M, FIG. 17N,FIG. 17O, FIG. 17P and FIG. 17Q compare peak areas for the SOPconfiguration and LBS configuration after initial injection when thesystems and columns had been used lightly. FIG. 17A, FIG. 17B, FIG. 17C,FIG. 17D, FIG. 17E, FIG. 17F, FIG. 17G, FIG. 17H, FIG. 17I, FIG. 17J,FIG. 17K, FIG. 17L, FIG. 17M, FIG. 17N, FIG. 17O, FIG. 17P and FIG. 17Qdisplay result for selected B vitamins, namely; B12, thiamine, MeB12,TPP, Aden B12, nicotinic acid, pyridoxine, folic acid, pyridoxamine,5MTHF, pyridoxal, pantothenic acid, riboflavin, PLP, nicotinamide,biotin and FMN, respectively, in accordance with the present disclosure.

FIG. 18A, FIG. 18B, FIG. 18C, FIG. 18D, FIG. 18E, FIG. 18F, FIG. 18G,FIG. 18H, FIG. 18I and FIG. 18J compare the chromatograms for the LBSconfiguration and the SOP configuration for selected B vitamins. FIG.18A, FIG. 18C, FIG. 18E, FIG. 18G and FIG. 18I display the chromatogramsfor the LBS configuration for flavin mononucleotide (FMN), thiamine(B₁), pyridoxal 5′-phosphate (PLP), pantothenic acid (B₅), and thiaminepyrophosphate (TPP) respectively, in accordance with the presentdisclosure. FIG. 18B, FIG. 18D, FIG. 18F, FIG. 18H and FIG. 18J displaythe chromatograms for the SOP configuration for flavin mononucleotide(FMN), thiamine (B₁), pyridoxal 5′-phosphate (PLP), pantothenic acid(B₅), and thiamine pyrophosphate (TPP) respectively, in accordance withthe present disclosure.

FIG. 19A, FIG. 19B, FIG. 19C, FIG. 19D, FIG. 19E, FIG. 19F, FIG. 19G,FIG. 19H, FIG. 19I, FIG. 19J, FIG. 19K and FIG. 19L show a comparison ofthe selected B vitamins LC-MS chromatograms obtained with the LBS andthe SOP configurations in a carry-over study. FIG. 19A, FIG. 19D, FIG.19G and FIG. 19J display LC-MS chromatograms of injections of a 10 ppmstandard solution for riboflavin, pyridoxal, 5-methyl-THF, andmethylcobalamin (Me B12), respectively, in accordance with the presentdisclosure. FIG. 19B, FIG. 19E, FIG. 19H and FIG. 19K display LC-MSchromatograms of blank injections obtained with the SOP configurationsfor riboflavin, pyridoxal, 5-methyl-THF, and methylcobalamin (Me B12),respectively, in accordance with the present disclosure. FIG. 19C, FIG.19F, FIG. 19I and FIG. 19L display LC-MS chromatograms of blankinjections obtained with the LBS configurations for riboflavin,pyridoxal, 5-methyl-THF, and methylcobalamin (Me B12), respectively, inaccordance with the present disclosure.

FIG. 20A, FIG. 20B, FIG. 20C and FIG. 20D display the chromatograms forthe LBS configuration and the SOP configuration for selected B vitaminsto compare carry-over for riboflavin. FIG. 20B and FIG. 20A show a blankinjection (FIG. 20A) right after an injection of a high concentrationstandard solution (10 ppm) (FIG. 20B) for LBS configuration. FIG. 20Cand FIG. 20D show a blank injection (FIG. 20D) right after an injectionof a high concentration standard solution (10 ppm) (FIG. 20C) for SOPconfiguration.

FIG. 21A, FIG. 21B, FIG. 21C and FIG. 21D display the chromatograms forthe LBS configuration and the SOP configuration for selected B vitaminsto compare carry-over for pyridoxal. FIG. 21B and FIG. 21A show a blankinjection (FIG. 21B) right after an injection of a high concentrationstandard solution (10 ppm) (FIG. 21A) for LBS configuration. FIG. 21Cand FIG. 21D show a blank injection (FIG. 21D) right after an injectionof a high concentration standard solution (10 ppm) (FIG. 21C) for SOPconfiguration.

FIG. 22A, FIG. 22B, FIG. 22C and FIG. 22D display the chromatograms forthe LBS configuration and the SOP configuration for selected B vitaminsto compare carry-over for 5-methyl-THF. FIG. 22B and FIG. 22A show ablank injection right after an injection of a high concentrationstandard solution (10 ppm) for LBS configuration. FIG. 22C and FIG. 22Dshow a blank injection right after an injection of a high concentrationstandard solution (10 ppm) for SOP configuration.

FIG. 23A, FIG. 23B, FIG. 23C and FIG. 23D display the chromatograms forthe LBS configuration and the SOP configuration for selected B vitaminsto compare carry-over for methyl-cobalamin. FIG. 23B and FIG. 23A show ablank injection (FIG. 23B) right after an injection of a highconcentration standard solution (10 ppm) (FIG. 20A) for LBSconfiguration. FIG. 23C and FIG. 23D show a blank injection (FIG. 23D)right after an injection of a high concentration standard solution (10ppm) (FIG. 23) for SOP configuration.

FIG. 24 displays the relative response factors for B vitamins on theLC-MS systems comparing the LBS configuration to the SOP configurationduring the LC-MS analysis of B vitamins.

FIG. 25A, FIG. 25B, FIG. 25C, FIG. 25D, FIG. 25E, FIG. 25F, FIG. 25G,FIG. 25H, FIG. 25I, FIG. 25J, FIG. 25K, FIG. 25L, FIG. 25M, FIG. 25N,FIG. 25O and FIG. 25P displays typical chromatograms of selected Bvitamins, namely; thiamine, PLP (Pyridoxal 5′-phosphate), pyridoxine,pyridoxal, pyridoxamine, nicotinic acid, pantothenic acid, nicotinamide,CN B12 (cyanocobalamine), MeB12 (methyl-cobalamine), 5MHTF(5-methyl-tetrahydrofolate), FA (folic acid), AdenB12(adenosyl-cobalamin), FMN (flavin mononucleotide), riboflavin, andbiotin, respectively, in accordance with the present disclosure.

FIG. 26A, FIG. 26B, FIG. 26C, FIG. 26D, FIG. 26E, FIG. 26F, FIG. 26G andFIG. 26H displays a typical chromatogram of selected B vitamins in anenergy drink sample. FIG. 26I, FIG. 26J, FIG. 26K, FIG. 26L, FIG. 26M,FIG. 26N, FIG. 26O, FIG. 26P, FIG. 26Q, FIG. 26R and FIG. 26S displays atypical chromatogram of selected B vitamins in a dietary supplementsample. FIG. 26A, FIG. 26B, FIG. 26C, FIG. 26D, FIG. 26E, FIG. 26F, FIG.26G and FIG. 26H displays a typical chromatogram of CN B12, FMN,riboflavin, pyridoxal, nicotinic acid, pantothenic acid, nicotinamide,respectively, in accordance with the present disclosure. FIG. 26I, FIG.26J, FIG. 26K, FIG. 26L, FIG. 26M, FIG. 26N, FIG. 26O, FIG. 26P, FIG.26Q, FIG. 26R and FIG. 26S displays a typical chromatogram of CN B12, FA(folic acid), FMN, riboflavin, biotin, thiamine, pyridoxine, pyridoxal,nicotinic acid, pantothenic acid, and nicotinamide, respectively, inaccordance with the present disclosure.

FIG. 27 displays the table of energy drink sample analysis with recoveryand repeatability.

FIG. 28 displays the table of dietary supplement sample analysis withrecovery and repeatability.

DETAILED DESCRIPTION

In general, the present disclosure is related to coating columns to haveLBS to increase peak intensity, peak shape, carry-over, and responsefactor by minimizing negative analyte/surface interactions that can leadto sample losses. The present disclosure addresses the problematicbinding of B vitamins and their vitamers on metallic surfaces ofchromatographic systems. For example, B vitamins can interact withstainless steel to reduce peak intensity.

In addition, coating the system to have a LBS minimizes uncertainty ofthe chromatographic system performance. Permanent passivation (or atleast semi-permanent passivation, i.e., useable lifetime of aconsumable) can be provided by the coating. For example, the system doesnot need to be passivated after each wash, and passivation does noteffectively diminish after each wash or flowing. Consequently, theanalyte detected using LC and a detector (e.g., MS, UV (for abundantspecies), etc.) can be depended upon as an accurate assessment of theanalyte present.

Analytes that interact with metal have often proven to be verychallenging to separate. The desire to have high pressure capablechromatographic systems with minimal dispersion has required that flowpaths decrease in diameter and be able to withstand increasingly highpressures at increasingly fast flow rates. As a result, the material ofchoice for chromatographic flow paths is often metallic in nature. Thisis despite the fact that characteristics of certain analytes, forexample, biomolecules, proteins, glycans, peptides, oligonucleotides,pesticides, bisphosphonic acids, anionic metabolites, and zwitterionslike amino acids and neurotransmitters, are known to have unfavorableinteractions, so called chromatographic secondary interactions, withmetallic surfaces.

The proposed mechanism for metal specific binding interactions requiresan understanding of the Lewis theory of acid-base chemistry. Pure metalsand metal alloys (along with their corresponding oxide layers) haveterminal metal atoms that have characteristics of a Lewis acid. Moresimply, these metal atoms show a propensity to accept donor electrons.This propensity is even more pronounced with any surface metal ionsbearing a positive charge. Analytes with sufficient Lewis basecharacteristics (any substance that can donate non-bonding electrons)can potentially adsorb to these sites and thus form problematicnon-covalent complexes. It is these substances that are defined asmetal-interacting analytes.

For example, B vitamins and their vitamers are capable of metalchelation. This interaction causes the B vitamins and their vitamers tobind to the flow path metals thus reducing the detected amounts of suchspecies, a particularly troublesome effect when B vitamins are the mostimportant analyte and have low concentration levels in the sample.

Other characteristics of analytes can likewise pose problems. Forexample, when the functional groups are ubiquitous in the sample, givingthe opportunity for cumulative analyte losses or undesirablechromatographic performance.

The inner surface of the flow path in LC systems and columns has arelatively small surface area as compared to the surface area of thepacking material in the chromatography column. Despite the small surfacearea, the inner surface in the flow path should not be overlooked as apotential source of unwanted interactions with target analytes. Stronginteractions between the metallic surfaces and analytes can lead tosurface adsorption of analytes which may result in poor peak shape,reduced or no peak response, and inaccurate results. There areworkarounds to address these issues, such as replacing thestainless-steel material with other materials, using additives in themobile phases to disturb the interactions, or coating the surface withstrong adsorbents prior to the analysis. These workarounds have theirlimitations and may negatively impact the chromatography performance andanalytical productivity.

For example, an alternative to using metal flow paths is to use flowpaths constructed from polymeric materials, such as polyether etherketone (PEEK). PEEK tubing, like most polymeric materials, is formed bymeans of an extrusion process. With polymeric resin, this manufacturingprocess can lead to highly variable internal diameters. Accordingly,PEEK column hardware yields unfavorable differences in the retentiontimes as can be observed from switching between one column and the next.Often, this variation can be a factor of three higher than a metalconstructed column. In addition, the techniques for fabricating polymerbased frits are not yet sufficiently developed to afford suitably ruggedcomponents for commercial HPLC columns. For example, commerciallyavailable PEEK frits tend to exhibit unacceptably low permeability.

One method of coating for LBS is the use of alkylsilyl coatings. In someaspects, the alkylsilyl coating acts a bioinert, low-bind coating tomodify a flow path to address flow path interactions with an analyte,such as a metal-sensitive analyte. That is, the bioinert, low-bindcoating minimizes surface reactions with the metal interacting compoundsand allows the sample to pass along a flow path without clogging,attaching to surfaces, or change in analyte properties. Thereduction/elimination of these interactions is advantageous because itallows for accurate quantification and analysis of a sample containing Bvitamin compounds or other metal-sensitive compounds. The coating whichcreates LBS along the flow path prevents/significantly minimizes analyteloss to the metallic surface walls, thereby reducing secondarychromatographic interactions.

LBS due to the coatings can provide an effective solution that mitigatesanalyte interactions with metal surfaces. Coatings used to create a LBScan include a highly cross-linked layer containing ethylene-bridgedsiloxane material that is similar to that of BEH particles. Relevantanalytes include organic acids, organophosphates, oligonucleotides,peptide, glycans, and phospholipids. B vitamins are water-solublevitamins that are essential for normal human metabolic and physiologicalfunctions. The routine testing of B vitamins is an important procedurefor food, beverage and nutraceutical quality control as well as fornutritional research. The present disclosure discussed LBS in theLC-MS/MS analysis of B vitamins.

FIG. 1 is a representative schematic of a chromatographic system/device100 that can be used to separate analytes, such as B vitamins and theirvitamers, in a sample. System 100 includes several components includinga fluid manager system 105 (e.g., controls mobile phase flow through thesystem), tubing 110 (which could also be replaced or used together withmicro fabricated fluid conduits), fluid connectors 115, frits 120, achromatography column 125, a sample injector 135 including a needle (notshown) to insert or inject the sample into the mobile phase, a vial, orsample container 130 for holding the sample prior to injection, and adetector 150, such as a mass spectrometer. Chromatography column 125 canbe a reversed phase column. Interior surfaces of the components of thechromatographic system/device 100 form a fluidic flow path that haswetted surfaces. Components of the fluidic flow path can have a lengthto diameter ratio of at least 20, at least 25, at least 30, at least 35or at least 40. The fluidic flow path can include wetted surfaces of anelectrospray needle (not shown).

At least a portion of the wetted surfaces can be LBS by coating with analkylsilyl coating to reduce secondary interactions. The coating cantailor the hydrophobicity of the wetted surfaces. The coating can beapplied by vapor deposition. As such, methods and devices of the presenttechnology provide the advantage of being able to use high pressureresistant materials (e.g., stainless steel) of a flow system, and thewetted surfaces of the fluidic flow path providing the appropriatehydrophobicity so deleterious interactions or undesirable chemicaleffects on the sample can be minimized.

In some examples, the coating of the flow path is non-binding withrespect to the analyte, such as a metal-sensitive compound (e.g., Bvitamins). Consequently, the analyte does not bind to the coating of theflow path.

The coating can be provided throughout the system from the tubing orfluid conduits 110 extending from the fluid manager system 105 all theway through to the detector 150. The coatings can also be applied tocomponents of the fluidic path. That is, one may choose to coat one ormore components or portions of a component and not the entire fluidicpath. For example, the internal portions of the column 125 and its frits120 and fluid connectors 115 can be coated whereas the remainder of theflow path can be left unmodified. Further, removable/replaceablecomponents can be coated. For example, the vial 130 containing thesample can be coated as well as frits 120.

In some examples, system 100 will need to be cleaned/cleared beforeevaluation begins in order to establish a baseline before beginningtests to determine suitability. Ensuring system 100 is at a baseline canhelp certify that there are no contaminants. It can also be used tovalidate a preparation process for system 100 after manufacturing ofsystem 100 is complete. For example, after system 100 is manufactured,method 300 of FIG. 3 (discussed below) can be used.

The flow path of the fluidic systems can be defined at least in part byan interior surface of tubing. The flow path of the fluidic systems canalso be described at least in part by an interior surface ofmicrofabricated fluid conduits. And the flow path of the fluidic systemscan be described as at least in part by an interior surface of a columnor at least in part by passageways through a frit. The flow path of thefluidic systems is also described at least in part by an interiorsurface of a sample injection needle or extending from the interiorsurface of a sample injection needle throughout the interior surface ofa column. In addition, the flow path can be described as extending froma sample container (e.g., a vial) disposed upstream of and in fluidiccommunication with the interior surface of a sample injection needlethroughout the fluidic system to a connector/port to a detector. In someexamples, all tubing, connectors, frits, membranes, sample reservoirs,and fluidic passageways along this fluidic path (wetted surfaces) arecoated.

In some examples, only the wetted surfaces of the chromatographic columnand the components located upstream of the chromatographic column areLBS, e.g., coated with an alkylsilyl coating, while wetted surfaceslocated downstream of the column are not coated. In other embodiments,all wetted surfaces are coated, including those surfaces downstream ofthe column. And in certain embodiments, wetted surfaces upstream of thecolumn, through the column, and downstream of the column to the entranceof inlet to the detector are coated. The coating can be applied to thewetted surfaces via vapor deposition. Similarly, the “wetted surfaces”of labware or other fluid processing devices may benefit from alkylsilylcoatings. The “wetted surfaces” of these devices not only include thefluidic flow path, but also elements that reside within the fluidic flowpath. For example, frits and/or membranes within a solid phaseextraction device come in contact with fluidic samples. As a result, notonly the internal walls within a solid phase extraction device, but alsoany frits/membranes are included within the scope of “wetted surfaces.”All “wetted surfaces” or at least some portion of the “wetted surfaces”can be improved or tailored for a particular analysis or procedure byincluding one or more of the coatings described herein. The term “wettedsurfaces” refers to all surfaces within a device (e.g., chromatographycolumn, chromatography injection system, chromatography fluid handlingsystem, frits, labware, solid phase extraction device, pipette tips,centrifuge tubes, beakers, dialysis chambers) that come into contactwith a fluid, especially a fluid containing an analyte of interest.

Further information regarding the coating and the deposition of coatingsin accordance with the present technology is available in US2019/0086371, which is hereby incorporated by reference.

A LBS configuration can include an alkylsilyl coating, as discussedherein. For example, a LBS configuration can include a C₂ coating, asdiscussed herein.

At least a portion of the wetted surfaces are coated with an alkylsilylcoating. The alkylsilyl coating is inert to at least one of the analytesin the sample. The alkylsilyl coating can have the Formula I:

R¹, R², R³, R⁴, R⁵, and R⁶ are each independently selected from(C₁-C₆)alkoxy, —NH(C₁-C₆)alkyl, —N((C₁-C₆)alkyl)₂, OH, OR^(A), and halo(i.e., a halogen, for example chloro). R^(A) represents a point ofattachment to the interior surfaces of the fluidic system. At least oneof R¹, R², R³, R⁴, R⁵, and R⁶ is OR^(A). X is (C₁-C₂₀)alkyl,—O[(CH₂)₂O]₁₋₂₀—, —(C₁-C₁₀)[NH(CO)NH(C₁-C₁₀)]₁₋₂₀-, or—(C₁-C₁₀)[alkylphenyl(C₁-C₁₀)alkyl]₁₋₂₀-.

When used in the context of a chemical formula, a hyphen (“-”) indicatesthe point of attachment. For example, when X is—[(C₁-C₁₀)alkylphenyl(C₁-C₁₀)alkyl]₁₋₂₀-, that means that X is connectedto SiR¹R²R³ via the (C₁-C₁₀)alkyl and connected to SiR⁴R⁵R⁶ via theother (C₁-C₁₀)alkyl. This applies to the remaining variables.

In one aspect, X in Formula I is (C₁-C₁₅)alkyl, (C₁-C₁₂)alkyl, or(C₁-C₁₀)alkyl. In some aspects, X in Formula I is methyl, ethyl, propyl,isopropyl, butyl, sec-butyl, iso-butyl, t-butyl, pentyl, hexyl, heptyl,nonyl, or decanyl. In other aspect, X in Formula I is ethyl or decanyl.

In one aspect, at least one of R¹, R², R³, R⁴, R⁵, and R⁶ is(C₁-C₆)alkoxy, e.g., ethoxy, wherein the values for X are described inFormula I or the preceding paragraph. In another aspect, at least two ofR¹, R², R³, R⁴, R⁵, and R⁶ is (C₁-C₆)alkoxy, e.g., ethoxy, wherein thevalues for X are described in Formula I or the preceding paragraph. Inanother aspect, at least three of R¹, R², R³, R⁴, R⁵, and R⁶ is(C₁-C₆)alkoxy, e.g., ethoxy, wherein the values for X are described inFormula I or the preceding paragraph. In another aspect, at least fourof R¹, R², R³, R⁴, R⁵, and R⁶ is (C₁-C₆)alkoxy, e.g., ethoxy, whereinthe values for X are described in Formula I or the preceding paragraph.In another aspect, at least five of R¹, R², R³, R⁴, R⁵, and R⁶ is(C₁-C₆)alkoxy, e.g., ethoxy, wherein the values for X are described inFormula I or the preceding paragraph.

In one aspect, at least one of R¹, R², R³, R⁴, R⁵, and R⁶ is halo, e.g.,chloro, wherein the values for X are described in Formula I or thepreceding paragraphs above. In another aspect, at least two of R¹, R²,R³, R⁴, R⁵, and R⁶ is halo, e.g., chloro, wherein the values for X aredescribed in Formula I or the preceding paragraphs above. In anotheraspect, at least three of R¹, R², R³, R⁴, R⁵, and R⁶ is halo, e.g.,chloro, wherein the values for X are described in Formula I or thepreceding paragraphs above. In another aspect, at least four of R¹, R²,R³, R⁴, R₅, and R⁶ is halo, e.g., chloro, wherein the values for X aredescribed in Formula I or the preceding paragraphs above. In anotheraspect, at least five of R¹, R², R³, R⁴, R₅, and R⁶ is halo, e.g.,chloro, wherein the values for X are described in Formula I or thepreceding paragraphs above.

In another aspect, R¹, R², R³, R⁴, R₅, and R⁶ are each methoxy orchloro.

In some embodiments, the alkylsilyl coating of Formula I is aorganosilica coating. In certain embodiments, the alkylsilyl coating ofFormula I is a hybrid inorganic/organic material that forms the wettedsurface or that coats the wetted surfaces.

The alkylsilyl coating of Formula I can have a contact angle of at leastabout 15°. In some embodiments, the alkylsilyl coating of Formula I canhave a contact angle of less than or equal to 30°. The contact angle canbe less than or equal to about 115°. In some embodiments, the contactangle of the alkylsilyl coating of Formula I is between about 150 toabout 90°, in some embodiments about 150 to about 105°, and in someembodiments about 150 to about 115°. For example, the contact angle ofthe alkylsilyl coating of Formula I can be about 0°, 5°, 10°, 15°, 20°,25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 85°, 90°,95°, 100°, 105°, 110°, or 115°.

The thickness of the alkylsilyl coating can be at least about 100 Å. Forexample, the thickness can be between about 100 Å to about 1600 Å. Thethickness of the alkylsilyl coating for Formal I can be about 100 Å, 200Å, 300 Å, 400 Å, 500 Å, 600 Å, 700 Å, 800 Å, 900 Å, 1000 Å, 1100 Å, 1200Å, 1300 Å, 1400 Å, 1500 Å or 1600 Å. The thickness of the alkylsilylcoating (e.g., a vapor deposited alkylsilyl coating) can be detectedoptically by the naked eye. For example, more opaqueness and colorationis indicative of a thicker coating. Thus, coatings with pronouncedvisual distinction are an embodiment of this technology. From thin tothick, the color changes from yellow, to violet, to blue, to slightlygreenish and then back to yellow when coated parts are observed underfull-spectrum light, such as sunlight. For example, when the alkylsilylcoating is 300 Å thick, the coating can appear yellow and reflect lightwith a peak wavelength between 560 and 590 nm. When the alkylsilylcoating is 600 Å thick, the coating can appear violet and reflect lightwith a peak wavelength between 400 and 450 nm. When the alkylsilylcoating is 1000 Å thick, the coating can appear blue and reflect lightwith a peak wavelength between 450 and 490 nm. See, e.g., Faucheu etal., Relating Gloss Loss to Topographical Features of a PVDF Coating,Published Oct. 6, 2004; Bohlin, Erik, Surface and Porous Structure ofPigment Coatings, Interactions with flexographic ink and effects ofprint quality, Dissertation, Karlstad University Studies, 2013:49.

In one aspect, the vapor deposited coating of Formula I is the productof vapor deposited bis(trichlorosilyl)ethane,bis(trimethoxysilyl)ethane, bis(trichlorosilyl)octane,bis(trimethoxysilyl)octane, bis(trimethoxysilyl)hexane, andbis(trichlorosilyl)hexane.

In some aspects, at least a portion of the wetted surfaces are coatedwith multiple layers of the same or different alkyslilyls, where thethickness of the alkylsilyl coatings correlate with the number oflayering steps performed (e.g., the number of deposited layers ofalkylsilyl coating on wetted surfaces (e.g., internal surfaces of thefluidic flow path of the chromatographic system/device or internalsurfaces or fluid interfacing/contacting surfaces of labware or otheranalytical devices, such as frits within a solid phase extraction devicetogether with interior walls of the solid phase extraction device). Inthis manner, increasingly thick bioinert coatings can be produced andtailored to achieve desirable separations.

The chromatographic device can have a second alkylsilyl coating indirect contact with the alkylsilyl coating of Formula I. The secondalkylsilyl coating has the Formula II

wherein R⁷, R⁸, and R⁹ are each independently selected from—NH(C₁-C₆)alkyl, —N[(C₁-C₆)alkyl]₂, (C₁-C₆)alkoxy, (C₁-C₆)alkyl,(C₁-C₆)alkenyl, OH, and halo; R¹⁰ is selected from (C₁-C₆)alkyl,—OR^(B), —[O(C₁-C₃)alkyl]₁₋₁₀O(C₁-C₆)alkyl, —[O(C₁-C₃)alkyl]₁₋₁₀OH andphenyl. (C₁-C₆)alkyl is optionally substituted with one or more halo.The phenyl is optionally substituted with one or more groups selectedfrom (C₁-C₃)alkyl, hydroxyl, fluorine, chlorine, bromine, cyano,—C(O)NH₂, and carboxyl. R^(B) is —(C₁-C₃)alkyloxirane,—(C₁-C₃)alkyl-3,4-epoxycyclohexyl, or —(C₁-C₄)alkylOH. The hashed bondto R¹⁰ represents an optional additional covalent bond between R¹⁰ andthe carbon bridging the silyl group to form an alkene, provided y is not0. y is an integer from 0 to 20.

In one aspect, y in Formula II is an integer from 1 to 15. In anotheraspect, y in Formula II is an integer from 1 to 12. In another aspect, yin Formula II is an integer from 1 to 10. In another aspect, y inFormula II is an integer from 2 to 9.

In one aspect R¹⁰ in Formula II is methyl and y is as described abovefor Formula II or the preceding paragraph.

In one aspect, R⁷, R⁸, and R⁹ in Formula II are each the same, whereinR¹⁰ and y are as described above. In one aspect, R⁷, R⁸, and R⁹ are eachhalo (e.g., chloro) or (C₁-C₆)alkoxy such as methoxy, wherein R¹⁰ and yare as described above.

In one aspect, y in Formula II is 9, R¹⁰ is methyl, and R⁷, R⁸, and R⁹are each ethoxy or chloro.

In one aspect, the coating of the formula II is n-decyltrichlorosilane,(3-glycidyloxypropyl)trimethoxysilane (GPTMS),(3-glycidyloxypropyl)trimethoxysilane (GPTMS) followed by hydrolysis,2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, trimethylchlorosilane,trimethyldimethyaminosilane, methoxy-polyethyleneoxy(3)silanepropyltrichlorosilane, propyltrimethoxysilane,(heptadecafluoro-1,1,2,2-tetrahydrodecyl)tris(dimethylamino)silane,(heptadecafluoro-1,1,2,2-tetrahydrodecyl)trischlorosilane,(heptadecafluoro-1,1,2,2-tetrahydrodecyl)trimethoxysilanevinyltrichlorosilane, vinyltrimethoxysilane, allyltrichlorosilane,2-[methoxy(polyethyleneoxy)3propyl]trichlorosilane,2-[methoxy(polyethyleneoxy)3propyl]trimethoxysilane, or2-[methoxy(polyethyleneoxy)3propyl]tris(dimethylamino)silane.

The alkylsilyl coating of Formula I and II can have a contact angle ofat least about 15°. In some embodiments, the alkylsilyl coating ofFormula I and II can have a contact angle of less than or equal to 105°.The contact angle can be less than or equal to about 115°. In otherembodiments, the contact angle can be less than or equal to about 90°.In some embodiments, the contact angle of the alkylsilyl coating ofFormula I and II is between about 150 to about 115°. For example, thecontact angle of the alkylsilyl coating of Formula I and II can be about0°, 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°,75°, 80°, 85°, 90°, 95°, 100°, 105°, 110°, or 115°.

The thickness of the multi-layered alkylsilyl coating can be at leastabout 100 Å. For example, the thickness can be between about 100 Å toabout 1600 Å. The thickness of the multi-layered alkylsilyl coating forFormal I can be about 100 Å, 200 Å, 300 Å, 400 Å, 500 Å, 600 Å, 700 Å,800 Å, 900 Å, 1000 Å, 1100 Å, 1200 Å, 1300 Å, 1400 Å, 1500 Å or 1600 Å.

In one aspect, the alkylsilyl coating of Formula I isbis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and thealkylsilyl coating of Formula II is(3-glycidyloxypropyl)trimethoxysilane. In another aspect, the alkylsilylcoating of Formula I is bis(trichlorosilyl)ethane orbis(trimethoxysilyl)ethane and the alkylsilyl coating of Formula II is(3-glycidyloxypropyl)trimethoxysilane followed by hydrolysis. In oneaspect, the alkylsilyl coating of Formula I is bis(trichlorosilyl)ethaneor bis(trimethoxysilyl)ethane and the alkylsilyl coating of Formula IIis n-decyltrichlorosilane. The alkylsilyl coating of Formula I can bebis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane and thealkylsilyl coating of Formula II can be trimethylchlorosilane ortrimethyldimethyaminosilane. In one aspect, the alkylsilyl coating ofFormula I is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane andthe alkylsilyl coating of Formula II is methoxy-polyethyleneoxy(3)propyl tricholorosilane or methoxy-polyethyleneoxy(3) propyltrimethoxysilane.

Exemplary coatings with their respective approximate thickness andcontact angle are provided in Table 1.

TABLE 1 Alternative Approximate Approximate Coating Thickness ContactVapor Deposited Material Abbreviation of Product Anglebis(trichlorosilyl)ethane or C₂ 500 Å  35° bis(trimethoxysilyl)ethaneAnnealed Annealed C₂ 500 Å  95° bis(trichlorosilyl)ethane orbis(trimethoxysilyl)ethane bis(trichlorosilyl)ethane or C₂C₁₀ 500 Å 105°bis(trimethoxysilyl)ethane as a first layer followed by n-decyltrichlorosilane as a second layer Annealed Annealed 500 Å 105°bis(trichlorosilyl)ethane or C₂C₁₀ bis(trimethoxysilyl)ethane as a firstlayer followed by annealed n-decyltrichlorosilane as a second layer

The first coating layer, C₂ shown below, is a layer according to FormulaI, described above.

C₂C₁₀ is an example of a coating of Formula I and a second layer ofFormula II. The structure of bis(trichlorosilyl)ethane orbis(trismethoxysilyl)ethane (C₂) is shown above. The structure of C₁₀ isshown below.

Alternatively, commercially available vapor deposition coatings can beused in the disclosed systems, devices, and methods, including but notlimited to Dursan® and Dursox® (both commercially available fromSilcoTek Corporation, Bellefonte, Pa.). The process for making isdescribed in U.S. application Ser. No. 14/680,669, filed on Apr. 7,2015, and entitled “Thermal Chemical Vapor Deposition Coated Article andProcess,” which claims priority to and benefit of U.S. ProvisionalApplication No. 61/976,789 filed Apr. 8, 2014. The contents of eachapplication are incorporated herein by reference in their entirety.

In some examples, coating the flow path includes uniformly distributingthe coating about the flow path, such that the walls defining the flowpath are entirely coated. In some embodiments, uniformly distributingthe coating can provide a uniform thickness of the coating about theflow path. In general, the coating uniformly covers the wetted surfacessuch that there are no “bare” or uncoated spots.

The coatings described above can be used to create LBS and can tailor afluidic flow path of a chromatography system for the separation of asample. The coatings can be vapor deposited. In general, the depositedcoatings can be used to adjust the hydrophobicity of internal surfacesof the fluidic flow path that come into contact with a fluid (i.e.wetted surfaces or surfaces coming into contact with the mobile phaseand/or sample/analyte). By coating wetted surfaces of one or morecomponents of a flow path within a chromatography system, a user cantailor the wetted surfaces to provide a desired interaction (i.e., alack of interaction) between the flow path and fluids therein (includingany sample, such as a sample containing B vitamins, within the fluid).

FIG. 2 is a flow chart illustrating method 200 for creating a LBS bytailoring a fluidic flow path for separation of a sample including Bvitamins and their vitamers. The method has certain steps which areoptional as indicated by the dashed outline surrounding a particularstep. Method 200 can start with a pretreatment step (205) for cleaningand/or preparing a flow path within a component for tailoring.Pretreatment step 205 can include cleaning the flow path with plasma,such as oxygen plasma. This pretreatment step is optional.

Next, an infiltration step (210) is initiated. A vaporized source of analkylsilyl compound is infiltrated into the flow path. The vaporizedsource is free to travel throughout and along the internal surfaces ofthe flow path. Temperature and/or pressure is controlled duringinfiltration such that the vaporized source is allowed to permeatethroughout the internal flow path and to deposit a coating from thevaporized source on the exposed surface (e.g., wetted surfaces) of theflow path as shown in step 215. Additional steps can be taken to furthertailor the flow path. For example, after the coating is deposited, itcan be heat treated or annealed (step 220) to create cross linkingwithin the deposited coating and/or to adjust the contact angle orhydrophobicity of the coating. Additionally or alternatively, a secondcoating of alkylsilyl compound (having the same or different form) canbe deposited by infiltrating a vaporized source into the flow path anddepositing a second or additional layers in contact with the firstdeposited layer as shown in step 225. After the deposition of eachcoating layer, an annealing step can occur. Numerous infiltration andannealing steps can be provided to tailor the flow path accordingly(step 230).

FIG. 3 provides a flow chart illustrating a method (300) of creating aLBS by tailoring a fluidic flow path for separation of a sampleincluding a analyte, such as B vitamins and their vitamers. The methodcan be used to tailor a flow system for use in isolating, separating,and/or analyzing B vitamins and their vitamers. In step 305, B vitaminsand their vitamers are assessed to determine polarity. Understanding thepolarity will allow an operator to select (by either look up table ormake a determination) a desired coating chemistry and, optionally,contact angle as shown in step 310.

In some embodiments, in addition to assessing the polarity of B vitaminsand their vitamers, the polarity of a stationary phase to be used toseparate the B vitamins and their vitamers (e.g., stationary phase to beincluded in at least a portion of the fluidic flow path) is alsoassessed. A chromatographic media (e.g., stationary phase) can beselected based on metal-sensitive compounds, e.g., B vitamins and theirvitamers, in the sample. Understanding the polarity of metal-sensitivecompounds (e.g., B vitamins and their vitamers) and the stationary phaseis used in certain embodiments by the operator to select the desiredcoating chemistry and contact angle in step 310. The components to betailored can then be positioned within a chemical infiltration systemwith environmental control (e.g., pressure, atmosphere, temperature,etc.) and precursor materials are infiltrated into the flow path of thecomponent to deposit one or more coatings along the wetted surfaces toadjust the hydrophobicity as shown in step 315. During any one ofinfiltration, deposition, and condition steps (e.g. annealing), coatingsdeposited from the infiltration system can be monitored and if necessaryprecursors and or depositing conditions can be adjusted if requiredallowing for fine tuning of coating properties.

The structures of the B vitamins and their bioactive vitamers are shownin FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, FIG. 4F, FIG. 4G, FIG.4H, FIG. 4I, FIG. 4J, FIG. 4K, FIG. 4L, FIG. 4M, FIG. 4N, and FIG. 4O.FIGS. 4A-4O show 18 compounds, namely; vitamin B₁₂, riboflavin (B₂),biotin, FMN (flavin mononucleotide), nicotinic acid (B₃), nicotinamide(B₃), nicotinamide adenine dinucleotide, pyridoxine (B₆), pyridoxamine(B₆), pyridoxal (B₆), PLP (Pyridoxal 5′-phosphate), thiamine (B₁), folicacid (B₉), 5MTHF (5-methyl-tetrahydrofolate), TPP (Thiaminepyrophosphate), and pantothenic acid (B₅), respectively.

Prior to any comparisons of coated column/hardware performance versusuncoated column/hardware performance for B vitamins and their vitamers,the following protocols were developed and used for sample preparationand analysis.

FIG. 5 displays the liquid chromatography conditions in the Associationof Official Analytical Chemists (AOAC) Official Method for simultaneousdetermination of total vitamins B1, B2, B3, and B6 and other nutrientsin matrices, such as infant formula and related nutritionals.

The present disclosure includes the simultaneous analysis of B vitamins.The examples included matrices of dietary supplements and energy drinkswith B vitamins. B vitamin complex dietary supplements can containmultiple B vitamins, including different vitamers. Some dietarysupplements include native, active, or coenzymated B vitamins, such asFMN and PLP. Energy drinks can contain vitamin B₁, B₂, B₃, B₅, B₆, B₇,B₉, B₁₂ and other ingredients (including natural product extracts).

Separate analyses are carried out for vitamins. For example, vitamin B₂is analyzed with the fluorometric method; vitamins B₃, B₅, B₉, and B₁₂are analyzed with a microbiological method; and vitamin B₆ with a liquidchromatography-fluorescence detector (LC-FLR) method. In contrast, thepresent disclosure analyzes B vitamins simultaneously, at least forsimple matrices, such as dietary supplements and beverages. For complexsamples, separate sample preparations for different B vitamins can berequired. After the B vitamins are extracted from the complex samplematrices, a single LC method can be used to determine the vitamins ofthe sample.

EXAMPLES

The examples studied the effects of the LBS on the analysis of Bvitamins. Comparisons were made between the SOP configuration (asdefined below) and the LBS configuration (as defined below) in peakarea, peak height, LOQ, peak shape, carry-over, and response factor

Configurations—(both commercially available from Waters TechnologiesCorporation, Milford, Mass., USA):

SOP: ACQUITY UPLC™ BEH™ C₁₈ Column in ACQUITY UPLC H Class Plus System

LBS: ACQUITY™ Premier BEH C₁₈ Column in ACQUITY Premier System

Significant differences were observed between the SOP and LBSconfigurations. As compared to the SOP configuration, using the LBSconfiguration produced better sensitivity and no carry-over.

Sample preparation for the samples included sample preparation fordietary supplements and for energy drinks. For dietary supplements, 50milligrams (recorded to 0.01 mg) of capsule content (fine powder), or 50μL of liquid dietary supplement, were dissolved in 200 ml water in ambervolumetric flasks. For powder samples, the samples were sonicated 5-10seconds. The samples stood in darkness for at least 2 hours to ensuredissolution. The sample solutions were then filtered with 0.45-micronGlass MicorFiber (GMF) syringe filter. The first 2 ml of the filtratewere discarded and then 4 ml of the filtrate were collected. Theanalysis solutions were prepared by mixing the sample filtrates withwater at different dilution ratios (95/100, 10/100, 1/100 v/vsample/total volume). Internal standard (IS) solution was also addedinto each analysis solution. For energy drinks, samples were sonicatedto remove carbonation and filtered through a 0.45 micron GMF syringemembrane filter. The filtrate was then diluted with water at differentdilution ratios (90/100, 2/100 v/v sample/total volume) prior toanalysis. IS solution was added into each analysis solution as well.

The following experimental conditions were used for the examples.

LC System Waters™ ACQUITY UPLC H Class Plus System (SOP configuration);ACQUITY Premier System (LBS configuration). The LBS configuration

LC Column Waters ACQUITY UPLC BEH C₁₈ Column (1.7 μm, 2.1×100 mm) (SOPconfiguration); ACQUITY Premier BEH C₁₈ Column (1.7 μm, 2.1×100 mm) (LBSconfiguration)

The SOP configuration is a system and column with stainless steel and nocoating. The LBS configuration is a system and column with stainlesssteel and a C₂ coating.

Mobile phases:

A: 20 mM ammonium formate in water (pH 5.0)

B: 100% methanol

Column temperature: 40° C.

Autosampler temperature: 5° C.

Injection volume: 2 μL

Gradient program:

Time Flow A B (min) (ml/min) (%) (%) Initial 0.35 99 1 0.50 0.35 99 12.50 0.35 92 8 5.00 0.35 10 90 6.00 0.35 10 90 6.10 0.35 99 1

MS system: Xevo TQ-S Micro System

MS system settings:

Polarity ES+ Capillary (kV) 1.4 Cone (V) 70 Source Temperature (° C.)150 Desolvation Temperature (° C.) 350 Cone Gas Flow (L/Hr) 100Desolvation Gas Flow (L/Hr) 650

FIG. 6A and FIG. 6B display the multiple reaction monitoring (MRM)transitions and MS detection parameters for 18 vitamins, including theadditional vitamins B₅, B₉, B₁₂ and biotin, that were used for the XevoTQ-S Micro MS System. Two LC system setups were used for comparison, oneincluded an ACQUITY Premier System and an ACQUITY Premier BEH C₁₈ (1.7m, 2.1×100 mm) Column with a C₂ coating (referred to as the LBSsetup/configuration), and the other one included an ACQUITY UPLC H ClassPlus System and an ACQUITY UPLC BEH C₁₈ (1.7 μm, 2.1×100 mm) Column(referred to as the SOP setup/configuration). An alkylsilyl coating withC₂ was used in the LBS setup while the stainless-steel parts were usedin the SOP setup. These two systems were coupled to the same Xevo TQ-SMicro MS System sequentially to minimize instrumental variables in thecomparison study.

FIG. 7 displays a table of calibration data, in accordance with examplesof the present disclosure. The table of calibration data includes foreach vitamin the limit of quantitation (LOQ) (ng/ml), range (ng/ml), R²,order of polynomial fitting, and internal standards.

FIGS. 8A-8R show comparison plots of the vitamin peak areas from 7replicate injections of a standard mix solution (at a concentration of 1μg/mL) in the SOP configuration and the LBS configuration. Peak areasfrom standard surface (SOP configuration) are plotted together withthose from LBS surface (LBS configuration). The B vitamins and vitamersinclude pyridoxine (FIG. 8A), thiamine (FIG. 8B), MeB12 (methylcobalamine) (FIG. 8C), TPP (Thiamine pyrophosphate) (FIG. 8D), Aden B12(adenosyl cobalamin)(FIG. 8E), nicotinic acid (FIG. 8F), riboflavin(FIG. 8G), nicotinamide (FIG. 8H), FMN (Flavin mononucleotide)(FIG. 8I),nicotinamide adenine dinucleotide (NAD)(FIG. 8J), B12 (FIG. 8K), folicacid (FIG. 8L), pyridoxamine (FIG. 8M), 5MTHF(50methyl-tetrahydrofolate)(FIG. 8N), pyridoxal (FIG. 8O), pantothenicacid (FIG. 8P), PLP (Pyridoxal 5′-phosphate)(FIG. 8Q), and biotin (FIG.8R). Increased responses of B vitamins were observed using the LBSconfiguration. LC systems were flushed and cleaned prior to thecomparison study. The data were obtained when the systems and columnswere new for both SOP configuration and the LBS configuration (no Bvitamins had ever been injected onto the systems). After initial LCequilibration and injections of blanks (solvent), consecutive injectionsof the same standard mix solution (concentrations were at 1 ppm, or 1μg/ml for each standard) were compared. The comparison was of LC-MSchromatographic peak areas of vitamin B₁, B₂, B₃, B₅, B₆, B₇, B₉, B₁₂and their vitamers in the initial 7 injections of the same standardsolution using the LBS configuration and the SOP configuration.

Higher peak intensities and larger peak areas were observed for majorityof the 18 vitamins using the LBS configuration. One can see that greateror the same responses (peak areas) were obtained in the LBSconfiguration for all 18 vitamins. Significant differences for TPP,Thiamine, FMN (flavin mononucleotide), PLP, and pantothenic acid wereobserved. The peak areas of these compounds from the LBS configurationwere significantly larger than those peak areas from the SOPconfiguration. TPP did not show any peak in the SOP configuration underthe conditions of the examples. Other compounds, such as cyanocobalamine(B12), methyl-cobalamine (MeB12), adenosyl-cobalamin (Aden B12),nicotinic acid, nicotinamide, pyridoxine, pyridoxaminde, pyridoxal,biotin, folic acid, and 5-methyl-tetrahydrofolate (5MTHF) show largerpeak areas in the LBS configuration than those in the SOP configuration.

FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E, FIG. 9F, FIG. 9G, and FIG.9H compare the chromatograms for the LBS configuration and the SOPconfiguration for flavin mononucleotide (FMN), thiamine (B₁), pyridoxal5′-phosphate (PLP), and pantothenic acid (B₅), respectively, inaccordance with the present disclosure. FIG. 9A, FIG. 9C, FIG. 9E, andFIG. 9G display the chromatograms for the LBS configuration for flavinmononucleotide (FMN), thiamine (B₁), pyridoxal 5′-phosphate (PLP), andpantothenic acid (B₅), respectively, in accordance with the presentdisclosure. FIG. 9B, FIG. 9D, FIG. 9F, and FIG. 9H display thechromatograms for the SOP configuration for flavin mononucleotide (FMN),thiamine (B₁), pyridoxal 5′-phosphate (PLP), and pantothenic acid (B₅),respectively, in accordance with the present disclosure. Vertical axesare the same scales for each compound comparison in FIG. 9A to FIG. 9H.The standard concentration was 1 ppm (μg/ml). The chromatograms of FIG.9A, FIG. 9C, FIG. 9E, and FIG. 9G show the positive effect the LBSconfiguration has on peak area (peak intensity).

FIG. 10A, FIG. 10B, FIG. 10C, and FIG. 10D are comparisons of LC-MSchromatograms of FMN (FIG. 10A), Thiamine (FIG. 10B), PLP (FIG. 10C),and Pantothenic acid (FIG. 10D) from the initial injection in the samestandard solution using LBS surfaces (LBS configuration) vs standardsurfaces (SOP configuration). FIGS. 10A-10D display that the peakintensities were significantly increased using the LBS configuration.Also, narrower peaks and less peak tailing was observed for the thiamine(FIG. 10B) and PLP (FIG. 10C) peaks in the LBS configuration. Theincrease in response in LC-MS/MS analysis of B vitamins with the LBSconfiguration was still evident after extended use of the LC-MS system.Peak intensities from standard surface (solid filled peaks) arenormalized to the peak intensities (not filled peak) from LBS.

FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, FIG. 11E, FIG. 11F, FIG. 11G,FIG. 11H, FIG. 11I, FIG. 11J, FIG. 11K, FIG. 11L, FIG. 11M, and FIG. 11Ncompare thiamine pyrophosphate (TPP) chromatograms from initial seveninjections for the SOP configuration and the LBS configuration, inaccordance with the present disclosure. FIG. 11A, FIG. 11B, FIG. 11C,FIG. 11D, FIG. 11E, FIG. 11F and FIG. 11G display chromatograms from7^(th), 6^(th), 5^(th), 4^(th), 3^(rd), 2^(nd) and 1^(st) injections forthe SOP configuration, respectively. FIG. 11H, FIG. 11I, FIG. 11J, FIG.11K, FIG. 11L, FIG. 11M and FIG. 11N display chromatograms from 7^(th),6^(th), 5^(th), 4^(th), 3^(rd) 2^(nd) and 1^(st) injections for the LBSconfiguration.

In the LBS configuration, as shown in FIG. 11H, FIG. 11I, FIG. 11J, FIG.11K, FIG. 11L, FIG. 11M and FIG. 11N, the TPP peak is gradually formedin shape after initial injections. In contrast, the SOP configuration,as shown in FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, FIG. 11E, FIG. 11Fand FIG. 11G, displays no TPP peak. FIG. 11H, FIG. 11I, FIG. 11J, FIG.11K, FIG. 11L, FIG. 11M and FIG. 11N displays how the LBS configurationhelps alleviate the issue of surface adsorption of TPP. The MS probe hasa metal surface that might contribute to the adsorption. The TPP peak inFIG. 11H, FIG. 11I, FIG. 11J, FIG. 11K, FIG. 11L, FIG. 11M and FIG. 11Nchanged during the consecutive injections. Without wishing to be boundby theory, the exposure history of the system and column to the TPP mayhave an effect in peak area and peak shape.

FIGS. 12A-12P compare the chromatograms of flavin mononucleotide (FMN)in standard solutions for the SOP configuration and the LBSconfiguration, in accordance with the present disclosure. FIG. 12A, FIG.12B, FIG. 12C, FIG. 12D, FIG. 12E, FIG. 12F, FIG. 12G and FIG. 12Hdisplay chromatograms for 10 ppm, 3 ppm, 1000 ppb, 300 ppb, 100 ppb, 30ppb, 10 ppb, and 3 ppb of flavin mononucleotide (FMN) concentrations,respectively, in standard solutions for the SOP configuration. FIG. 12I,FIG. 12J, FIG. 12K, FIG. 12L, FIG. 12M, FIG. 12N, FIG. 12O and FIG. 12Pdisplay chromatograms for 10 ppm, 3 ppm, 1000 ppb, 300 ppb, 100 ppb, 30ppb, 10 ppb, and 3 ppb of flavin mononucleotide (FMN) concentrations,respectively, in standard solutions for the LBS configuration.

The standard concentration ranged from 3 ppb to 10 ppm. For the SOPconfiguration, as shown in FIGS. 12A-12H, the limit of quantitation(LOQ) was approximately 1000 ppb. For the LBS configuration, as shown inFIGS. 12I-12P, the LOQ was lower at approximately 100 ppb. In addition,peak shape was symmetric for the LBS configuration of FIGS. 12I-12P andthe peak shape for the SOP configuration of FIGS. 12A-12H, showedtailing. A comparison of the results of FIGS. 12A-12H to FIGS. 12I-12PI,illustrates that using the LBS configuration enhanced sensitivity andpeak shape.

FIGS. 13A-13P compare the chromatograms of pyridoxal 5′-phosphate (PLP)in standard solutions for the SOP configuration and the LBSconfiguration, in accordance with the present disclosure. FIG. 13A, FIG.13B, FIG. 13C, FIG. 13D, FIG. 13E, FIG. 13F, FIG. 13G and FIG. 13Hdisplay chromatograms for 10 ppm, 3 ppm, 1000 ppb, 300 ppb, 100 ppb, 30ppb, 10 ppb, and 3 ppb of pyridoxal 5′-phosphate (PLP) concentrations,respectively, in standard solutions for the SOP configuration. FIG. 13I,FIG. 13J, FIG. 13K, FIG. 13L, FIG. 13M, FIG. 13N, FIG. 13O and FIG. 13Pdisplay chromatograms for 10 ppm, 3 ppm, 1000 ppb, 300 ppb, 100 ppb, 30ppb, 10 ppb, and 3 ppb of pyridoxal 5′-phosphate (PLP) concentrations,respectively, in standard solutions for the LBS configuration.

Thus, FIGS. 13A-13H display chromatograms using the SOP configuration;whereas FIGS. 13I-13P display chromatograms using the LBS configuration.The standard concentration ranged from 3 ppb to 10 ppm. For the SOPconfiguration, as shown in FIGS. 13A-13H, the limit of quantitation(LOQ) was approximately 1000 ppb. For the LBS configuration, as shown inFIGS. 13I-13P, the LOQ was lower at approximately 300 ppb. In addition,peak shape was narrower for the LBS configuration of FIGS. 13I-13P thanthe SOP configuration of FIGS. 13A-13H. A comparison of the results ofFIGS. 13A-13H to FIGS. 13I-13P, illustrates that using the LBSconfiguration enhanced sensitivity and peak shape.

FIGS. 14A-14P compare the chromatograms of thiamine in standardsolutions for the SOP configuration (FIGS. 14A-14H) and the LBSconfiguration (FIG. 14I-FIG. 14P), in accordance with the presentdisclosure. FIG. 14A, FIG. 14B, FIG. 14C, FIG. 14D, FIG. 14E, FIG. 14F,FIG. 14G and FIG. 14H display chromatograms for 10 ppm, 3 ppm, 1000 ppb,300 ppb, 100 ppb, 30 ppb, 10 ppb, and 3 ppb of thiamine concentrations,respectively, in standard solutions for the SOP configuration. FIG. 14I,FIG. 14J, FIG. 14K, FIG. 14L, FIG. 14M, FIG. 14N, FIG. 14O and FIG. 14Pdisplay chromatograms for 10 ppm, 3 ppm, 1000 ppb, 300 ppb, 100 ppb, 30ppb, 10 ppb, and 3 ppb of thiamine concentrations, respectively, instandard solutions for the LBS configuration.

The standard concentration ranged from 3 ppb to 10 ppm. For the SOPconfiguration, as shown in FIGS. 14A-14H, the limit of quantitation(LOQ) was approximately 1000 ppb. For the LBS configuration, as shown inFIGS. 14I-14P, the LOQ was lower at approximately 300 ppb. In addition,peak shape was narrower and showed less tailing for the LBSconfiguration of FIGS. 14I-14P than the SOP configuration of FIGS.14A-14H. A comparison of the results of FIGS. 14A-14H to FIGS. 14I-14P,illustrates that using the LBS configuration enhanced sensitivity andpeak shape.

FIGS. 15A-15P compare the chromatograms of pantothenic acid in standardsolutions for the SOP configuration (FIGS. 15A-15H) and the LBSconfiguration (FIGS. 15I-15P), in accordance with the presentdisclosure. FIG. 15A, FIG. 15B, FIG. 15C, FIG. 15D, FIG. 15E, FIG. 15F,FIG. 15G and FIG. 15H display chromatograms for 10 ppm, 3 ppm, 1000 ppb,300 ppb, 100 ppb, 30 ppb, 10 ppb, and 3 ppb of pantothenic acidconcentrations, respectively, in standard solutions for the SOPconfiguration. FIG. 15I, FIG. 15J, FIG. 15K, FIG. 15L, FIG. 15M, FIG.15N, FIG. 15O and FIG. 15P display chromatograms for 10 ppm, 3 ppm, 1000ppb, 300 ppb, 100 ppb, 30 ppb, 10 ppb, and 3 ppb of pantothenic acidconcentrations, respectively, in standard solutions for the LBSconfiguration.

The standard concentration ranged from 3 ppb to 10 ppm. For the SOPconfiguration, as shown in FIGS. 15A-15H, the limit of quantitation(LOQ) was approximately 30 ppb. For the LBS configuration, as shown inFIGS. 15I-15P, the LOQ was lower at approximately 10 ppb. In addition,peak shape was slightly narrower for the LBS configuration of FIGS.15I-15P than the SOP configuration of FIGS. 15A-15H. A comparison of theresults of FIGS. 15A-15H to FIGS. 15I-15P, illustrates that using theLBS configuration enhanced sensitivity and peak shape.

FIGS. 16A-16P compare the chromatograms of nicotinic acid in standardsolutions for the SOP configuration (FIGS. 16A-16H) and the LBSconfiguration (FIGS. 16I-16P), in accordance with the presentdisclosure. FIG. 16A, FIG. 16B, FIG. 16C, FIG. 16D, FIG. 16E, FIG. 16F,FIG. 16G and FIG. 16H display chromatograms for 10 ppm, 3 ppm, 1000 ppb,300 ppb, 100 ppb, 30 ppb, 10 ppb, and 3 ppb of nicotinic acidconcentrations, respectively, in standard solutions for the SOPconfiguration. FIG. 16I, FIG. 16J, FIG. 16K, FIG. 16L, FIG. 16M, FIG.16N, FIG. 16O and FIG. 16P display chromatograms for 10 ppm, 3 ppm, 1000ppb, 300 ppb, 100 ppb, 30 ppb, 10 ppb, and 3 ppb of nicotinic acidconcentrations, respectively, in standard solutions for the LBSconfiguration.

The standard concentration ranged from 3 ppb to 10 ppm. For the SOPconfiguration, as shown in FIGS. 16A-16H, the limit of quantitation(LOQ) was approximately 50 ppb. For the LBS configuration, as shown inFIGS. 16I-16P, the LOQ was lower at approximately 30 ppb. A comparisonof the results of FIGS. 16A-16H to FIGS. 16I-16P, illustrates that usingthe LBS configuration enhanced sensitivity.

FIGS. 17A-17Q, compare peak areas for the SOP configuration and LBSconfiguration after initial injection when the systems and columns hadbeen used lightly. Peak areas from standard surface (SOP configuration)are plotted side by side with those from LBS surface (LBSconfiguration). FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D, FIG. 17E, FIG.17F, FIG. 17G, FIG. 17H, FIG. 17I, FIG. 17J, FIG. 17K, FIG. 17L, FIG.17M, FIG. 17N, FIG. 17O, FIG. 17P and FIG. 17Q are similar comparison tothe comparison of FIG. 8A-FIG. 8R but repeated after the initialinjections. For FIG. 17A-FIG. 17Q, the comparison of the configurationsis completed after 31 injections of standard solutions at variousconcentrations ranging from 3 ppb to 10 ppm. The comparison wasconducted with a standard mix solution at 0.3 ppm, or 0.3 μg/ml. Thecolumns and systems of FIG. 17A-FIG. 17Q were conditioned with blankinjections, and no cleaning or flushing was done. Observations for FIG.17A-FIG. 17Q were similar to the observations of FIG. 8A-FIG. 8R. Largedifferences for TPP, Thiamine, FMN (flavin mononucleotide), PLP, andpantothenic acid were observed. The peak areas of these compounds fromthe LBS configuration were significantly larger than those peak areasfrom the SOP configuration. The TPP and thiamine analysis did not showany peak in the SOP configuration under these conditions. Othercompounds, such as cyanocobalamine (B12), methyl-cobalamine (MeB12),adenosyl-cobalamin (Aden B12), nicotinic acid, nicotinamide, pyridoxine,pyridoxaminde, pyridoxal, and biotin show larger peak areas for the LBSconfiguration than those in the SOP configuration.

FIG. 18A, FIG. 18B, FIG. 18C, FIG. 18D, FIG. 18E, FIG. 18F, FIG. 18G,FIG. 18H, FIG. 18I and FIG. 18J compare the chromatograms for the LBSconfiguration and the SOP configuration for flavin mononucleotide (FMN),thiamine (B₁), pyridoxal 5′-phosphate (PLP), pantothenic acid (B₅), andthiamine pyrophosphate (TPP) respectively, in accordance with thepresent disclosure. FIG. 18A, FIG. 18C, FIG. 18E, FIG. 18G and FIG. 18Idisplay the chromatograms for the LBS configuration for flavinmononucleotide (FMN), thiamine (B₁), pyridoxal 5′-phosphate (PLP),pantothenic acid (B₅), and thiamine pyrophosphate (TPP) respectively, inaccordance with the present disclosure. FIG. 18B, FIG. 18D, FIG. 18F,FIG. 18H and FIG. 18J display the chromatograms for the SOPconfiguration for flavin mononucleotide (FMN), thiamine (B₁), pyridoxal5′-phosphate (PLP), pantothenic acid (B₅), and thiamine pyrophosphate(TPP) respectively, in accordance with the present disclosure.

Vertical axes are the same scales. The standard concentration was 0.3ppm (μg/ml). The chromatograms of FIG. 18A, FIG. 18C, FIG. 18E, FIG. 18Gand FIG. 18I show the positive effect the LBS configuration has on peakarea (peak intensity) after initial injections. FIG. 18A-FIG. 18Jprovide a similar comparison to the comparison of FIG. 9A-FIG. 9H butrepeated after the initial injections. For FIG. 18A-FIG. 18J, theinitial injections were 31 injections of standard solutions at variousconcentrations ranging from 3 ppb to 10 ppm.

To summarize the results for sensitivity, the use of LBS in LC systemand column helps to increase the peak area (intensity), reduce peaktailing and peak width for B vitamins, which result in better detectionsensitivity. The peak areas of thiamine, TPP, FMN, PLP, pantothenic acidwere significantly increased in the LBS configuration than thoseobtained in the SOP configuration at concentration 1 ppm (1 μg/ml)during the initial injections of B vitamin standard mix solutions onto anew column in a flush and cleaned LC system. Marginal increase in peakarea was observed for cyanocobalamin, methyl-cobalamin,adenosyl-cobalamin, nicotinic acid, nicotinamide, pyridoxine,pyridoamine, pyridoxal, biotin. No difference in peak area was observedfor riboflavin, NAD, folic acid, and 5-Methyl-THF. A second comparison(FIG. 17A-FIG. 17Q and FIG. 18A-FIG. 18J) of chromatograms of a standardmix solution at 0.3 ppm of each standard from both SOP and LBSconfigurations showed a similar observation. The second comparison wasconducted when the system and column had been lightly used (31injections of standard solutions at various concentration). As a result,higher detection sensitivities for some B vitamins were obtained in theLBS configuration. No significant detrimental effect was observed for Bvitamins in the LBS configuration.

The increased responses of the LBS configuration lead to highersensitivities in the LC-MS analysis of the B vitamins. The limit ofquantification (LOQ) in both the SOP configuration and LBS configurationwas estimated using the same standard solutions at concentrationsranging from 3 ng/mL to 10 μg/mL. Large improvement in LOQ (3-10 timesimprovement) was achieved for FMN, thiamine, PLP and pantothenic acid inthe LBS system setup (Table 1).

TABLE 1 Estimated LOQ values for selected B vitamins LOQ LOQ (ng/mL)improvement SOP LBS in LBS than Compound configuration configuration SOPsetup FMN 1000 100 10 times  Thiamine 1000 300 3 times PLP 1000 300 3times Pantothenic acid 30 10 3 timesRegarding carry-over, the LBS configuration minimizes interactionsbetween the B vitamins and the stainless-steel inner surface of the flowpath in LC system and column, and reduces the risk of carry-over thatoften occurs in the LC analysis of B-group of vitamins.

FIG. 19A, FIG. 19B, FIG. 19C, FIG. 19D, FIG. 19E, FIG. 19F, FIG. 19G,FIG. 19H, FIG. 19I, FIG. 19J, FIG. 19K and FIG. 19L show a comparison ofthe selected B vitamins LC-MS chromatograms obtained with the LBS andthe SOP configurations in a carry-over study. FIG. 19A, FIG. 19D, FIG.19G and FIG. 19J display LC-MS chromatograms of injections of a 10 ppmstandard solution for riboflavin, pyridoxal, 5-methyl-THF, andmethylcobalamin (Me B12), respectively, in accordance with the presentdisclosure. FIG. 19B, FIG. 19E, FIG. 19H and FIG. 19K display LC-MSchromatograms of blank injections obtained with the SOP configurationsfor riboflavin, pyridoxal, 5-methyl-THF, and methylcobalamin (Me B12),respectively, in accordance with the present disclosure. FIG. 19C, FIG.19F, FIG. 19I and FIG. 19L display LC-MS chromatograms of blankinjections obtained with the LBS configurations for riboflavin,pyridoxal, 5-methyl-THF, and methylcobalamin (Me B12), respectively, inaccordance with the present disclosure.

For FIG. 19A-FIG. 19L, carry-over was not observed using the LBSconfiguration. Comparison of LC-MS chromatograms of blank injectionsobtained with the LBS configuration and with SOP configuration rightafter the injections of a 10 ppm standard solution for riboflavin (FIGS.19A-19C), pyridoxine (FIGS. 19D-19F), 5-methyl-THF (FIGS. 19G-19I), andmethylcobalamin (FIGS. 19J-19L). Residual peaks were found in the blankinjection on the SOP configuration for riboflavin, pyridoxine,5-methyl-tetrahydrofolate, and methylcobalamin while no residual peakwas found on the LBS configuration. The LBS configuration reduces therisk of carry-over in the LC-MS analysis for B vitamins.

FIG. 20A, FIG. 20B, FIG. 20C and FIG. 20D display the chromatograms forthe LBS configuration and the SOP configuration for selected B vitaminsto compare carry-over for riboflavin. FIG. 20B shows a blank injectionright after an injection of a high concentration standard solution (10ppm) (FIG. 20A) for a LBS configuration. FIG. 20D shows a blankinjection (FIG. 20D) right after an injection of the high concentrationstandard solution (10 ppm) (FIG. 20C) for a SOP configuration. Forriboflavin, the blank injection in the SOP configuration showed aresidue peak at about 0.1% of previous peak's area, and the blankinjection showed no residue peak in the LBS configuration.

FIG. 21A, FIG. 21B, FIG. 21C and FIG. 21D display the chromatograms forthe LBS configuration and the SOP configuration for selected B vitaminsto compare carry-over for pyridoxal. FIG. 21B shows a blank injectionright after an injection of a high concentration standard solution (10ppm) (FIG. 21A) for a LBS configuration. FIG. 21D shows a blankinjection right after an injection of the high concentration standardsolution (10 ppm) (FIG. 21C) for the SOP configuration. For pyridoxal,the blank injection in the SOP configuration showed a residue peak atabout 0.1% of previous peak's area, and the blank injection showed noresidue peak in the LBS configuration.

FIG. 22A, FIG. 22B, FIG. 22C and FIG. 22D display the chromatograms forthe LBS configuration and the SOP configuration for selected B vitaminsto compare carry-over for 5-methyl-THF. FIG. 22B shows a blank injectionright after an injection of a high concentration standard solution (10ppm) (FIG. 22A) for the LBS configuration. FIG. 22D shows a blankinjection right after an injection of the high concentration standardsolution (10 ppm) (FIG. 22C) for the SOP configuration. For5-methyl-THF, the blank injection in the SOP configuration showed aresidue peak at about 0.1% of previous peak's area, and the blankinjection showed no residue peak in the LBS configuration.

FIG. 23A, FIG. 23B, FIG. 23C and FIG. 23D display the chromatograms forthe LBS configuration and the SOP configuration for selected B vitaminsto compare carry-over for methyl-cobalamin. FIG. 23B shows a blankinjection right after an injection of a high concentration standardsolution (10 ppm) (FIG. 23A) for the LBS configuration. FIG. 23D show ablank injection right after an injection of the high concentrationstandard solution (10 ppm) (FIG. 23C) for the SOP configuration. Formethyl-cobalamin, the blank injection in the SOP configuration showed aresidue peak at about 0.03% of previous peak's area, and the blankinjection showed no residue peak in the LBS configuration.

No significant carry-over issue exists for B vitamins in both the LBSconfiguration and the SOP configuration. Small residue peaks forriboflavin, pyridoxal, 5-methyl-THF and methyl-cobalamin were observedin blank injections following an injection of a high concentrationstandard mix solution (10 ppm) for the SOP configuration. The residuepeaks were about 0.1% or less of the previous 10 ppm standard peakareas. In contrast, for the LBS configuration, there was no residue peakfound in the blank injection for B vitamins. The use of LBS in LCsystems and columns helps to reduce the potential carry-over issue for Bvitamins.

The effect of LBS on the analysis of B vitamins includes the responsefactors, which were calculated using the calibration data inconcentration range close to their LOQ. The response factors are morecomprehensive than a single concentration level comparison in term ofcomparing the response in two configurations for the LBS configurationand the SOP configuration. That is, comparison at a fix concentrationlevel may not reflect the complete picture since the LOQ and thelinearity for the B vitamins are not the same. Some B vitamins are bestfitted with quadratic curves. If the concentration level is too low ortoo high, the difference observed at that concentration would appearlarger or smaller. Response factors for B vitamins on the LBSconfiguration and the SOP configuration (the same Xevo TQ-S Micro Systemcoupled to two LC systems) were compared during extensive use (up to 170injections).

FIG. 24 displays the relative response factors for B vitamins on theLC-MS systems comparing the LBS configuration to the SOP configurationduring the LC-MS analysis of B vitamins. Calibration standard solutionsthat were injected during the B vitamin analysis were used for theresponse factors, which were calculated based on the calibration datapoints in low concentration ranges above their LOQs. Data was collectedduring an extensive use of the system, in which total of 170 injectionsof standard solutions, sample solutions, and blanks were made on bothconfigurations (LBS and SOP). The response factors were calculated fromthe calibration data points obtained at different periods. To obtainrelative response factors, the response factors from the LBSconfiguration were normalized to those from the SOP configuration foreach compound. The average values were plotted with the error barrepresents the ±RSD in the response factor (n=4). After extensive use,the LBS configuration still exhibits higher response factors (>150%) forsome B vitamins: such as thiamine, biotin, nicotinic acid, and 5MTHF.

FIG. 25A, FIG. 25B, FIG. 25C, FIG. 25D, FIG. 25E, FIG. 25F, FIG. 25G,FIG. 25H, FIG. 25I, FIG. 25J, FIG. 25K, FIG. 25L, FIG. 25M, FIG. 25N,FIG. 25O and FIG. 25P displays typical chromatograms of selected Bvitamins, namely; thiamine, PLP (Pyridoxal 5′-phosphate), pyridoxine,pyridoxal, pyridoxamine, nicotinic acid, pantothenic acid, nicotinamide,CN B12 (cyanocobalamine), MeB12 (methyl-cobalamine), 5MHTF(5-methyl-tetrahydrofolate), FA (folic acid), AdenB12(adenosyl-cobalamin), FMN (flavin mononucleotide), riboflavin, andbiotin, respectively, in accordance with the present disclosure. ForFIG. 25A-FIG. 25P, the chromatograms are of standard solution at 3 ppm(μg/ml).

FIG. 26A, FIG. 26B, FIG. 26C, FIG. 26D, FIG. 26E, FIG. 26F, FIG. 26G andFIG. 26H displays a typical chromatogram of selected B vitamins in anenergy drink sample. FIG. 26I, FIG. 26J, FIG. 26K, FIG. 26L, FIG. 26M,FIG. 26N, FIG. 26O, FIG. 26P, FIG. 26Q, FIG. 26R and FIG. 26S displays atypical chromatogram of selected B vitamins in a dietary supplementsample. FIG. 26A-FIG. 26H display a typical chromatogram of CN B12, FMN,riboflavin, pyridoxal, nicotinic acid, pantothenic acid, nicotinamide,respectively, in accordance with the present disclosure. FIG. 26I-FIG.26S display a typical chromatogram of CN B12, FA (folic acid), FMN,riboflavin, biotin, thiamine, pyridoxine, pyridoxal, nicotinic acid,pantothenic acid, and nicotinamide, respectively, in accordance with thepresent disclosure. As compared to the typical examples of FIG. 25A-FIG.25P, the present disclosure provides chromatograms with higher response,better peak shapes, and no carry-over. The analysis of B vitaminsreceives the benefit of higher sensitivity, higher accuracy andprecision, and enhanced robustness for the analysis of B vitamins.

FIG. 27 displays the table of energy drink sample analysis with recoveryand repeatability.

FIG. 28 displays the table of dietary supplement sample analysis withrecovery and repeatability. For FIG. 27 and FIG. 28, the results wereobtained on a LBS configuration system.

The use of LBS in LC system and column helps to increase the peak area(intensity), reduce peak tailing and peak width for B vitamins, whichresult in a better detection sensitivity. In addition, the use of LBS inLC system and column helps to reduce the potential carry-over issue forB vitamins.

The present disclosure highlights the benefits of using the LBSconfiguration for the LC-MS/MS analysis of B-group vitamins. Thebenefits include sharper and more symmetric peak shapes, greater peakareas, increased peak area (intensity), reduced peak tailing and peakwidth for B vitamins, which result in a better detection sensitivity. Inaddition, the use of LBS in LC system and column helps to reduce thepotential carry-over issue for B vitamins. These improvements helpanalysts to quantify B vitamins at lower concentrations, with higheraccuracy and precision, and enhanced robustness.

The above aspects and features of the present technology providenumerous advantages over the prior art. In some embodiments, there arenumerous benefits incorporating the coating through the column (and insome embodiments through the entire fluidic pathway from samplereservoir to the detector) to define a LBS (e.g., an organosilica coatedsurface). For example, the present disclosure shows the benefits ofreducing secondary interactions, which includes positively impactingchromatographic performance in terms of band broadening, peak tailing,and/or recovery which can then help increase resolution, peak capacity,and/or quantitative accuracy of B vitamins and their vitamers.

1-21. (canceled)
 22. A method of separating and analyzing ametal-sensitive sample, the method comprising: injecting themetal-sensitive sample into a chromatographic system, wherein thechromatographic system comprises a metallic flow path with a low-bindsurface coating; flowing the metal-sensitive sample through thechromatographic system; separating the metal-sensitive sample, whereinthe metal-sensitive sample comprises one or more B vitamin and/or Bvitamin vitamer; and analyzing the separated sample by passing theseparated metal-sensitive sample through a mass spectrometer.
 23. Themethod of claim 22, wherein the sample comprises two or more of Bvitamins and/or B vitamin vitamers, and wherein analyzing the separatedsample comprises simultaneously analyzing the two or more of B vitaminsand/or B vitamin vitamers to determine the B vitamins and/or B vitaminvitamers present in the sample.
 24. The method of claim 22, whereinprior to injecting the metal-sensitive sample into the chromatographicsystem, extracting the metal-sensitive sample from a sample matrix orsample matrices.
 25. The method of claim 24, wherein extracting themetal-sensitive sample from a sample matrix or sample matrices comprisesseparate sample preparations for different B vitamins.
 26. The method ofclaim 22, wherein flowing the metal-sensitive sample through thechromatographic system comprises using a single liquid chromatographymethod to determine the B vitamins and/or B vitamin vitamers.
 27. Themethod of claim 26, wherein the single liquid chromatography methodcomprises using liquid chromatography-mass spectrometry (LC/MS).
 28. Themethod of claim 27, wherein the liquid chromatography-mass spectrometry(LC/MS) is a tandem quadrupole mass spectrometer (LC-MS/MS).
 29. Themethod of claim 22, wherein the B vitamins or B vitamin vitamerscomprise at least one of riboflavin (B₂), flavin mononucleotide (FMN),biotin, nicotinic acid (B₃), nicotinamide (B₃), nicotinamide adeninedinucleotide (NAD), pyridoxine (B₆), pyridoxal (B₆), pyridoxal5′-phosphate (PLP), thiamine (B₁), thiamine pyrophosphate (TPP), folicacid (B₉), pantothenic acid (B₅), or 5-methyl-tetrahydrofolate (5MTHF).30. The method of claim 22, wherein the sample is a dietary supplementor an energy drink.
 31. A method of separating and analyzing ametal-sensitive sample, the method comprising: injecting themetal-sensitive sample into a chromatographic system, wherein thechromatographic system comprises a metallic flow path with a coating;flowing the metal-sensitive sample through the chromatographic system;separating the metal-sensitive sample, wherein the metal-sensitivesample comprises two or more B vitamins and/or B vitamin vitamers; andsimultaneously analyzing the two or more B vitamins and/or B vitaminvitamers of the separated sample.
 32. The method of claim 31, whereinthe coating is a low-bind surface coating.
 33. The method of claim 31,wherein analyzing the separated sample comprises passing the separatedmetal-sensitive sample through a mass spectrometer.
 34. The method ofclaim 31, wherein prior to injecting the metal-sensitive sample into thechromatographic system, extracting the metal-sensitive sample from asample matrix or sample matrices.
 35. The method of claim 34, whereinextracting the metal-sensitive sample from a sample matrix or samplematrices comprises separate sample preparations for different B vitaminsand/or B vitamin vitamers.
 36. The method of claim 31, wherein flowingthe metal-sensitive sample through the chromatographic system comprisesusing a single liquid chromatography method to determine the B vitaminsand/or B vitamin vitamers.
 37. The method of claim 36, wherein thesingle liquid chromatography method comprises using liquidchromatography-mass spectrometry (LC/MS).
 38. The method of claim 37,wherein the liquid chromatography-mass spectrometry (LC/MS) is a tandemquadrupole mass spectrometer (LC-MS/MS).
 39. The method of claim 31,wherein the B vitamins or B vitamin vitamers comprise riboflavin (B₂),flavin mononucleotide (FMN), biotin, nicotinic acid (B₃), nicotinamide(B₃), nicotinamide adenine dinucleotide (NAD), pyridoxine (B₆),pyridoxal (B₆), pyridoxal 5′-phosphate (PLP), thiamine (B₁), thiaminepyrophosphate (TPP), folic acid (B₉), pantothenic acid (B₅), or5-methyl-tetrahydrofolate (5MTHF).